| Nanopore-based sensing has developed into a promising single molecule technique over the past two decades.Due to its some merits such as simplicity,low cost,lable-free and high sensitivity,it has been widely used in DNA sequencing,protein conformational analysis,probing enzyme kinetics,biological macromolecular interaction and single-molecule detection of various substances including nucleic acids,polypeptides,protein,metal ions,small organic molecules and so on.Among the group of nanopore,the transmembrane toxin ?-Hemolysin(?HL)protein is most often studied because of its good structural reproducibility and easily genetic engineering modification.However,selective detection of small molecules at the single-molecule level is still difficult because the low signal resolution and weak anionic selectivity of the wild-type ?HL towards small molecules.Herein,we designed and synthesized two functionalized cyclodextrin derivatives with positively charged groups,which are utilized as both an adaptor and the recognition site in the mutant ?HL protein nanopore for selective recognition and sensing of some biologically important anions at single-molecule level.The main research contents are included as follows:1.Designing an imidazolium-functionalized ?-cyclodextrin adaptor for simultaneous recognition of phosphate anions in a nanopore.We develop a stochastic nanopore sensing method for simultaneous determination of phosphate-containing anions using an imidazolium-functionalized ?-cyclodextrin(?CDIM)as an adaptor.The sensing approach is based on the hydrogen-bonding and/or electrostatic interactions between the target anions and ?CDIM lodged in the lumen of mutant ?-hemolysin(?HL),which resulted in differential current modulations in the nanopore.It was demonstrated that this system could excellently discriminate pyrophosphate(PPi)in the presence of other phosphate-containing compounds,such as dihydrogen phosphate(Pi),tripolyphosphate(PPPi)and even the equally charged adenosine triphosphate(ATP)under physiological conditions.With this method,pyrophosphate(PPi)could be selectively and sensitively detected at very low concentration with LOD of 6.3 n M in the range of 14 n M–4.6 ?M.More importantly,the adaptor-based sensor was successfully applied to the determination of PPi in human plasma samples,which suggests that our proposed method is promising and reliable in monitoring the level of phosphate-containing anions for diagnostic purposes and may have great application value and development potential in real-time DNA sequencing.2.Design and synthesis of per-6-quaternaryammonium-?-cyclodextrin(p-QABCD)for single molecule detection of L-cysteine.Many methods for the determination of L-cysteine have been reported previously.However,a great incentive still exists to develop a fast and simple analytical method to detect L-cysteine selectively and sensitively,especially at single-molecule level.In this paper,we used a newly synthesized per-6-quaternary-ammonium-?-cyclodextrin(p-QABCD),as molecular adaptor in the mutant ?HL protein(M113R)7 nanopore for recognizing target molecules.By recording and analyzing the change of characteristic current blocking signal of DTNB before and after chromogenic reaction of Ellman reagent(DTNB)with L-cysteine in the nanopore,L-cysteine can be indirectly quantitative detected at single-molecule level.Besides,we have also compared the intermediate product during the synthesis of p-QABCD,per-6-tertiaryammonium-?-cyclodextrin(p-TABCD).Due to its relative hydrophobicity,p-TABCD can enhance the binding ability of nanopore towards DTNB molecule and simultaneously distinguish the reactant DTNB and the product TNBA when it is lodged in the lumen of mutant ?HL-(M113R)7 pore.Therfeore,this functionalized cyclodextrin adaptor is expected to further improve the recognition performance of nano-channel for L-cysteine and other mercapto small molecules,and provides a new possibility to investigate the mechanism of DTNB with thiol groups in nano-size confinement. |
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